Bacteria determination method



BACTERIA DETERMINATION METHOD Filed April 9, 1962 DIPTO LINE 15W pl/Z Q //5' I 1/9 J20 (We, rce

United States Patent This invention relates to a bacteria determination device and method, and, more particularly, to a determination of bacteria in urine.

The importance of testing for asymptomatic bacteriuria has been described in a number of investigations Kass 3 has shown that the determ nation of asymptomatic bacteriuria is of special importance during pregnancy. The need for repetitive screening for bacteriuria throughout pregnancy. The need for repetitive screening for bacteriuria throughout pregnancy has also been reported by Kaitz. The object of this invention is to provide a product and method for a simple quantitative test for bacteriuria.

The second most common infection in man is an infection of the urinary tract (the most common being that of the upper respiratory tract). It has been found that 10% of all pregnant women develop urinary tract infections. However, unlike upper respiratory tract infections, urinary tract infections tend to be without symptoms and for this reason they are not discovered by the physician until considerable damage to the kidney has occurred. After the damage has occurred, then albumin, red blood cells, and white blood cells are to be found in the urine. Presently, the methods for enumeration of bacteria are too complicated to be used in the physicians ofi'ice and for most hospitals.

The general requirements for a screening procedure are reproducible methods of removing a specimen and uniformly distributing it on an agar plate. This has not been possible through the enzymatic reduction of such redox dyes as resazurin and methylene blue, which previously had been used to estimate the number of bacteria in milk. The inadequacy of dyes for providing a simple quantitative test for bacteriuria stems from the fact that reducing substances in the urine altered the initial concentration of the oxidized form of the dye which changes the time required for reduction. Further, such bacteria as Pseudomonas, Aerobacter, Eschericha, Proteus and Staphylococci were found to vary significantly in their activity toward the indicators. Still further, three to five hours were required for a visible color change to occur when 10 bacteria per milliliter were used in the test. During such a period of incubation, bacterial multiplication occurred which obscured the initial differences in metabolic activity.

Contemporary methods such as the wire loop method which employ colony counting, were found to be unsatisfactory because dilution of the urine specimen, removal of a loopful of the diluted urine and careful streaking of the specimen to insure uniform distribution of the colonies are techniques requiring too much time and skill for the test to be satisfactory as a screening procedure.

1 R. A. McDonald, H. Levitin, G. K. Mallory and E. H. Kass Relation between pyelonephritis and bacterial counts in the urine. New Eng. J. Med. 256 922, 1957.

G. Jackson, K. Peter Poirier and H. G. Grieble: Concepts of pyelonephritis: Experience with renal biopsies and long term clinical observations. Annals of Internal Medicine 47 11651l83, 1957.

E. H. Kass: The role of asymptomatic bacteriuria in the pathogenesis of pyelonephritis. p. 399-411, Biology of Pyelonephritis, Henry Ford Hospital International Symposium. Little, Brown and Company, 1959.

A. L. Kaitz and E. W. Hodder: Bacteriuria and pyelonephritis of pregnancy. A prospective study of 016 pregnant women. The New Eng. J. Med. 265: 667-672, 1961.

Patented Sept. 15, 1964 'ice In one embodiment of the invention, I employ a strip of fibrous material, such as filter paper, for first immersing the same partway in urine and thereafter developing a foot-like part for streaking of the culture medium.

The invention will be described in conjunction with an illustrative embodiment in the accompanying drawing, in which- FIG. 1 is a side elevational view of the strip prior to immersion;

FIG. 2 is a view similar to FIG. 1 but in which the strip has a changed configuration to develop a foot-like part after immersion and which is useful in the abovementioned streaking procedure;

FIG. 3 is a top plan view of the device as seen in FIG. 2;

FIG. 4 is a plan view of a culture plate which has been streaked by devices such as seen in FIG. 3; and

FIG. 5 is a plan View of a modified form of the device.

Referring now to the drawing, the numeral 10 designates generally a fibrous strip such as a filter paper, out into a strip A" wide and 3" long. The strip 10 is equipped with a line of weakness 11 (such as perforation) which permits the development of an integral footlike portion 12 (see FIG. 2). The development of the foot-like portion is achieved by pressing the end 13 of the strip 10 successively into the culture medium as at 14, 15, 16 and 17. In particular, it will be noted that the streaking achieved through the contact at 17 was substantially complete.

In the illustration given, I find that the incipient fold line 11 is advantageously placed about one-half inch from the end 13. Also, there may be a printed indicia such as at 18 to inform the physician how far to dip the strip 10. Further, I find that to use the smallest area of agar possible and still get reproducible results, the area of the foot-like portion 12 should lie in the range of 0.05 sq. in. to 0.04 sq. in. Also, I find it advantageous not to impregnate the strip 10 prior to immersion in the urine and that optimum results are obtained when a relatively dense filter paper is used. I find that the dense filter papers will give higher counts than soft absorbent papers, and bacteria that grow in clusters or chains also produce higher colony counts than bacteria that grow singly. Although it is possible to increase the colony count by using dense filter paper or by gelatin impregnation of the paper, it is preferred to use unimpregnated Carl Schleicher & Schuell Co. 470 or 470-A filter papers for the screening test because the discrepancy between counts obtained with bacteria occurring in clusters or chains and those occurring singly is believed to provide a more accurate reflection of the actual number of bacteria present. Such papers are dense and the number of fibers per square inch is of the order of 1,630,000.

Variables in the procedure such as the time of application of the paper to the agar, and the time between dip ping the strip and application to the agar were measured. Variations in time for 20 seconds to 3 minutes for the procedure produced insignificant changes in the number of colonies transferred.

A standard curve relating the number of bacteria per milliliter to the number of colonies per filter strip was prepared by diluting 18 hour cultures of E. coli, Aerobacter aerogenes, Staphylococci aureus, Proteus vulgaris, Pseudomonas aeruginosa and Streptococcus faecalia in 0.85% saline. The dilutions were prepared so that concentrations near 10,000, 50,000, 100,000, 500,000 and 1,000,000 bacteria per milliliter would be obtained. A standard plate count was used to determine the number of bacteria present in each of the dilutions. The filter paper strips were dipped into each of the dilutions and the foot of the strip placed on an agar plate. After incubation, the number of colonies in the area inoculated sneepsa 3 were counted. The data for this example is shown in Table 1 below:

The experiment was repeated with dilution of the bacteria in filter-sterilized urine without significant differences occurring in the counts obtained.

I have found that the filter paper test described is simple and provides a more accurate picture of the bacterial count than does the plate count. In theory, each colony in a plate count is produced by one bacterium. However, this is true only when the bacteria used in the plate count occur singly. A cluster of staphylococci or a chain of streptococci will produce only one colony and are therefore counted as one bacterium, although 16 or 20 bacteria may occur in the cluster or chain. For this reason, the data obtained with the unimpregnated filter paper strips is closer to the true number of bacteria present than the standard plate count, since the larger the number of bacteria in the cluster or chain the greater will be the number of colonies transferred to the agar plate by the filter strip.

Yow 5 has shown that in certain cases of staphylococci urinary tract infections the bacterial concentration of the urine is less than bacteria/ml. These lower concentrations of bacteria per milliliter were determined by plate count and may be a result of the inherent error found in plate counts of microorganisms which occur in clusters or chains.

The principal alternative to the use of the plate count is microscopic counts which are free from the error described above and also give information as to the type of bacteria present. However, the microscopic count will be accurate only if an exact volume of sample is spread over an exact area so that each microscopic field represents an aliquot of the sample. Routine examination of sediment for bacteria is subject to enormous errors since none of the above variables are controlled. In addi tion, the removal of the bacteria from the urine by centrifugation using ordinary speeds is not quantitative and is an additional source of error. In contrast to this, the inventive test provides a simple quantitative procedure for the detection of asymptomatic bacteriuria and the test also has value in following the effectiveness of treatment of urinary tract infections.

In summary, the test is eifectively performed when the material used is absorbent so that drops of liquid are not transferred to the agar plate, the material consisting of fibers packed tightly together so that each fiber acts as a small inoculation needle so that with the more fibers, the more bacteria will be transferred. I find that the E. M. Yow, O. T. Monzon, E. M. Ory and .T. C. Brennan: The Microfiora of the Urinary Tract, p. 391-398, Biology of Pyelonephritis, Henry Ford Hospital International Symposium, Little, Brown and Company, 1959.

strip of filter paper must be straight when it enters the urine specimen and then bent to form the inoculating surface when pressed against the agar. The reason for this is that a bend in the paper will cause it to pick up more of the specimen. The more the bend, the more material will be picked up; that is, the strip would act like a spoon when bent. This makes it impossible to quantitate the procedure. In order to provide this type of action, it is necessary that the paper be weakened as by perforation or indentation so that it bends under light pressure. Further, the filter paper possesses sufficient rigidity and Wet strength to permit its use as an applicator. The test strips may be also advantageously utilized in testing additionally for pH in albumin, these variables constituting important tests in urinalysis. This is achieved, as can be seen by reference to FIG. 5, by impregnating an area as at 119 of the paper with a pH indicator or indicators covering the range of between about 5.5 to about 7.5. Again, the strip 110 is equipped with the line of weakness 1311 providing a foot-like area 112 having an edge 113. In addition, another area as at is impregnated with tetra bromophenol sulfon phthalein (0.05%) dissolved in alcohol. To 100 cc. of the alcoholic solution of the dye is added 4 gramsof tartaric acid. This solution detects albumin by changing color from yellow to blue.

While in the foregoing specification I have set down a detailed description of the invention for the purpose of illustration thereof, many variations in the details herein given may be made by those skilled in the art without departing from the spirit and scope of the invention.

I claim: In the quantitative testing for asymptomatic bacteriuria, the steps of:

partially immersing lengthwise in urine a generally elongated rectangular filter strip while the same is in straight form, said strip having previously been provided with a transverse line of weakness adjacent the inserted end to define a foot-like portion having fibers densely packed together, said portion having an area of about 0.05-0.40 square inch and constituting a minor part of the strip, successively applying the immersed end of the strip to a culture medium to develop an angular relation between the foot-like portion and the remainder of the strip while simultaneously streaking the culture medium, and counting the bacteria colonies developed in the culture medium.

References Cited in the file of this patent UNITED STATES PATENTS 2,464,155 Russell et a1. Mar. 8, 1949 2,904,474 Forg Sept. 15, 1959 2,985,288 Reich May 23, 1961 3,001,915 Fonner Sept. 26, 1961 OTHER REFERENCES Frobisher: Fundamentals of Microbiology, 5th ed., W. B. Saunders Company (1953), page 201 (copy in Div. 

